Re-generation of cytotoxic γδT cells with distinctive signatures from human γδT-derived iPSCs

Summary For a long time, ex vivo-expanded peripheral-blood-derived γδT cell (PBγδT)-based immunotherapy has been attractive, and clinical trials have been undertaken. However, the difficulty in expanding cytotoxic γδT cells to an adequate number has been a major limitation to the efficacy of treatment in most cases. We successfully re-generated γδT cells from γδT cell-derived human induced pluripotent stem cells (iPSCs). The iPSC-derived γδT cells (iγδTs) killed several cancer types in a major histocompatibility complex (MHC)-unrestricted manner. Single-cell RNA sequencing (scRNA-seq) revealed that the iγδTs were identical to a minor subset of PBγδTs. Compared with a major subset of PBγδTs, the iγδTs showed a distinctive gene expression pattern: lower CD2, CD5, and antigen-presenting genes; higher CD7, KIT, and natural killer (NK) cell markers. The iγδTs expressed granzyme B and perforin but not interferon gamma (IFNγ). Our data provide a new source for γδT cell-based immunotherapy without quantitative limitation.


INTRODUCTION
Gamma delta T (gdT) cells attack various types of cancer cells in a major histocompatibility complex (MHC)-unrestricted manner (Wrobel et al., 2007). Therefore, peripheral blood-derived gdT cell (PBgdT)-based immunotherapy has received attention, and clinical trials have been undertaken (Kobayashi et al., 2010). Because the proportion of gdT cells in adult peripheral blood mononuclear cells amounts to only a few percent or less (Aljurf et al., 2002), gdT cells need to be expanded by stimulants ex vivo for clinical use (Khan et al., 2021). However, in most cases, the difficulty in expanding cytotoxic gdT cells derived from peripheral blood to an adequate number has been a major limitation to the efficacy of the treatment (Wada et al., 2014). Furthermore, PBMC-derived gdT cells can be expanded enough for them to be used for autologous adoptive immunotherapy, but not enough for them to be used for allogenic mass-produced immunotherapeutic modalities.
Induced pluripotent stem cell (iPSC) technology may be able to overcome these limitations and enable us to realize off-the-self allogenic gdT cell-based therapy, which has several advantages over autologous gdT cells therapy: (1) the ex vivo expansion rate of PBMC-derived gdT cells varies widely from donor individual to individual; thus, ex vivoexpanded autologous PBgdT cell therapy cannot be applied to all patients; (2) patients have to wait for the expansion of autologous cells but not for off-the shelf allogenic cells; and (3) an autologous approach would be associated with high costs. iPSCs have infinite proliferation ability: they show logarithmic growth for at least 100 days and $1,028-fold expansion during 100 days ($10,000-fold/2 weeks) (Nakagawa et al., 2014). To generate gdT cells from human iPSCs (hiPSCs), the favorable cell of origin of the iPSC is a gdT cell, because T cell receptor (TCR) gene rearrangement is theoretically retained throughout the process of reprogramming and differentiation. Indeed, abT cells differentiated from abT cell-derived hiPSCs reportedly retained parental abTCR gene rearrangement (Nishimura et al., 2013;Vizcardo et al., 2013), and re-generated abT cells from iPSCs showed an antigen-specific cytotoxicity to cancer cells (Nishimura et al., 2013).
Previously we successfully established hiPSC lines from peripheral blood-derived gdT cells with a simple and clinically applicable method (Watanabe et al., 2018). These gdT cell-derived iPSCs (gdT-iPSCs) were demonstrated to be able to differentiate into CD34(+)CD43(+) hematopoietic progenitor cells. However, it has not yet been clarified whether the gdT-iPSCs can differentiate into gdT cells that can kill various types of cancer in an MHC-unrestricted manner. In a previous report by Zeng et al. (2019), the authors reported the generation of ''mimetic gdT cells'' endowed with natural killer (NK) receptors from gdT cell-derived iPSCs and designated them as gdNKT cells. However, the cells do not match any type of physiologically existing, authentic, or bona fide lymphocyte, including gdT cells, and should be categorized as cells resulting from an aberrant characteristic of lymphocytes derived from iPSCs, or abnormal cells. Previous studies have demonstrated that such abnormal cells can be derived from pre-rearranged TCR-carrying pluripotent stem cells or hematopoietic progenitors, and such iPSCs reportedly gave rise to abnormal  Ascorbic acid EPO (legend on next page) T cells expressing TCR, an NK cell marker NK1.1, and CD8aa (Vizcardo et al., 2018). Similarly, the resultant cells generated by Zeng et al. also express TCR, NK cell molecules, and CD8aa, suggesting the possibility that the cells might be mere "abnormal T cells" that had previously been known to be derived from pre-rearranged TCR-carrying pluripotent stem cells through unphysiological processes but not cells with any potential clinical utility.
Accordingly, if the gdT-iPSCs could differentiate into gdT cells, whether the molecular signatures of the re-generated resultant gdT cells from the gdT-iPSCs are identical to some subset of authentic gdT cells or absolutely artificial and unnatural cells should be revealed.
In this study, we successfully re-generated gdT cells from gdT-iPSCs. The iPSC-derived gdT cells (igdTs) exhibited cytotoxicity against several cancer cell lines in an MHC-unrestricted manner. We identified distinctive molecular signatures of igdTs and clarified that the igdTs were identical to a minor subset of ex vivo-expanded PBgdT cells. Our data provide a new source for gdT cell-based immunotherapy without quantitative limitations.

RESULTS
Re-differentiation of gdT-iPSCs into gdT cells For re-differentiation into gdT cells, we used two gdTderived hiPSC lines from different donors: 62B3, which was established in our previous report (Watanabe et al., 2018), and 121-3, which was newly established in this study. We confirmed that both iPSC clones expressed undifferentiated markers (NANOG, OCT3/4, and SOX2) at protein levels ( Figure S1A) and mRNA levels ( Figure S1B) and that the Sendai virus vector used for the introduction of reprogramming factors had been removed ( Figure S1B). An in vitro embryoid body (EB)-mediated differentiation experiment showed that they could differentiate into three germ layers ( Figure S1C). In a Q-band analysis, karyotype abnormality was not observed ( Figure S1D). Genomic PCR to examine the rearrangement at the TCRG and TCRD gene locus showed Vg9-to-JP and Vd2-to-JD1 recombination ( Figure S1E). These data verified that the two lines (62B3 and 121-3) are gdT-derived iPSCs (gdT-iPSC) that carry Vg9Vd2-TCR genes.
Next, we re-differentiated these gdT-iPSCs into gdT cells according to previously reported protocols (Kutlesa et al., 2009;Watanabe et al., 2018) with slight modifications shown in Figure 1A. On day 10, we confirmed the induction of cells positive for both CD34 and CD43 ( Figure 1B upper panels), the subset of which was shown to be hematopoietic progenitor cells (HPCs) (Timmermans et al., 2009). At this time point, no cells expressed CD7, CD3, or gdTCR ( Figure 1B middle and lower panels). From day 10, the derivatives of gdT-iPS were co-cultured with OP9/ N-DLL1 feeder cells, which have been commonly used for differentiation of HPCs into T lymphocytes (Kutlesa et al., 2009;Schmitt et al., 2004). Thereafter, the expression of CD34 gradually became negative, and cells positive for CD7, a pre-lymphoid and mature T cell marker (Ohishi et al., 2002;Timmermans et al., 2009), increased ( Figure 1B middle panels).
On day 30, the expression of CD3 was clearly positive, while the expression of gdTCR was still weak ( Figure 1B lower panels). To differentiate not only nonadherent differentiated cells but also more immature cells adhering to the feeder (Jing et al., 2010), we collected all cells, including feeder cells, and transferred them into a feeder-free dish. We then started gdTCR stimulation with (E)-4-Hydroxy-3methyl-but-2-enyl diphosphate (HMBPP) ( Figure 1A), which is a metabolite in a non-mevalonate pathway and which is known to activate Vg9Vd2 T cells (Nerdal et al., 2016). Although some feeder cells adhered to the new dish, they peeled off after several days (data not shown). Seven to 10 days after the start of gdTCR stimulation, we found cell aggregations with phase-contrast microscopy ( Figure 1A), and most of the cells became clearly positive for both CD3 and gdTCR ( Figure 1B lower panels), suggesting the maturation of the cells to gdT cells progressed. We confirmed reproducibility of the differentiation to CD3(+) gdTCR(+) cells from two gdT-iPSC lines ( Figure 1C). We named the resultant cells igdTs. At day 40 of differentiation, we obtained up to 3 3 10 5 igdTcells from 2 3 10 3 iPSCs. Even after the induction of igdT, CD3-negative cells still existed. Although it was unclear what type of cells the CD3-negative cells were, they were at least negative for CD56 and CD335, which are known markers of NK cells ( Figure S2A).

Monoclonal gdTCR expression in igdTs
Next, we examined whether the igdTs expressed a monoclonal gdTCR, as theoretically expected. In contrast to CD3-positive cells in peripheral blood mononuclear cells (PBMCs) stimulated with HMBPP for 1 week being composed of both gdTCR-positive and abTCR-positive cells, CD3-positive igdTs contained no abTCR-positive cells (Figure 2A). Genomic PCR to detect TCRG and TCRD genes showed that Vg9-to-JP and Vd2-to-JD1 recombination in gdT-iPSCs was retained in igdTs ( Figure 2B). Furthermore, we performed an analysis of the TCR g and TCR d repertoire of CD3(+) gdTCR(+) cells sorted from the igdTs as well as HMBPP-stimulated PBgdTs. The results of the amino acid sequences in the CD3R lesion, which guarantee reliability only for amino acid sequences with a frequency of more than 1%, demonstrated that PBgdTs consisted of more than 25 clones (Figure 2C right panels; Table S1), whereas igdTs consisted of only a single clone ( Figure 2C left panels; Table S2)).
These data indicated that we successfully re-generated monoclonal g9d2 T cells via gdT-iPSCs.

Cytotoxicity of igdTs against cancer cell lines
A key advantage of gdT cells for cancer immunotherapy is that one type of gdT cell is applicable for various types of cancer in a human leukocyte antigen (HLA)-unrestricted manner (Wrobel et al., 2007). We therefore evaluated the toxicity of the igdTs against four types of cancer cell lines, including two non-solid tumor (Jurkat cells [an acute T cell leukemia cell line] and K562 cells [a chronic myelogenous leukemia cell line]) and two solid tumors (Huh-7 cells [a hepatocellular carcinoma cell line] and SW480 cells [a colorectal adenocarcinoma cell line]). We confirmed that these cell lines have different HLA types from the two iPSC lines used in this study (Table 1).
First, we labeled Jurkat cells with carboxyfluorescein diacetate succinimidyl ester (CFSE) as target cells, co-cultured with the igdTs as effector cells at an effector (E):target (T) ratio of 2:1 and stained these cells with 7aminoactinomycin-D (7-AAD) to identify dead cells, followed by flow cytometry (FCM). The no-effector condition showed that only approximately 5% of Jurkat cells were dead ( Figure 3A left panel). In contrast, the derivatives of iPSCs showed obvious cytotoxicity; co-culture with effector cells for 1 day resulted in cell death of approximately half of the target cells, regardless of whether or not CD3 and gdTCR co-positive cells were sorted ( Figure 3A middle and right panels). Accordingly, we decided to use unsorted igdTs as effector cells for the subsequent cytotoxicity assays to avoid cellular damage caused by sorting. We confirmed the reproducibility of cytotoxicity using a gdT-iPSC line, 62B3-derived igdTs, generated in eight independent differentiation experiments. All the experiments showed the cytotoxicity of igdTs toward Jurkat cells (Figure 3B), although the magnitude of efficacy varied from experiment to experiment. Another gdT-iPSC line, 121-3derived igdTs, also showed cytotoxicity toward Jurkat cells ( Figure S3A).
To assess the cytotoxicity of igdTs toward solid tumor cells, we observed the co-culture of 62B3 gdT-iPSC linederived igdTs with GFP-Huh-7 cells by time-lapse imaging. After 12 h of co-culture, the areas of GFP-Huh-7 cells decreased to 28.7%, 30.1%, and 64.5% of those in noeffector control culture in three independent experiments ( Figures 3C and 3D). Moreover, we were able to catch igdTs coming into contact with GFP-Huh-7 cells and peeling off as time went on (Video S1). The change in the area of GFP-Huh-7 cells each hour is shown in Figure S3B. Similarly, co-culture of CFSE-labeled SW480 and the derivatives of a gdT-iPSC line, 62B3, resulted in a decrease in the areas of SW480 cells to 38.6%, 64.4%, and 66.0% of those of the no-effector control at 16 h in three independent experiments ( Figures 3E and 3F). Another gdT-iPSC line, 121-3-derived igdTs, also showed cytotoxicity toward Huh-7 cells ( Figure S3C) and SW480 cells ( Figure S3D).
Next, to confirm the cytotoxicity of purified-igdTs, we co-cultured CD3-MACS-purified igdT cells and tumor cells and quantified the tumor toxicity at an E:T ratio of 2:1 at 12 h using xCELLigence. Against Huh-7 cells, the purified igdT and PBgdT cells showed no significant difference in cytotoxicity ( Figure 3G). Moreover, because gdT cells reportedly express NK receptor molecules, such as NKG2D (Rincon-Orozco et al., 2005), we performed a cytotoxicity assay using K562 cells, which are known to be killed by NK cells, as target cells. igdT cells and peripheral blood-derived NK (PBNK) cells showed similar cytotoxic activity against K562 cells, with no significant difference ( Figure 3H).
These data demonstrated that the igdTs have cytotoxicity for at least four different types of cancer cells in an HLA-unrestricted manner. Moreover, we were able to catch igdTs coming into contact with GFP-Huh-7 cells and peeling off as time went on (Video S1).

Mode of action of igdTs
We next performed several experiments to obtain insight into the mode of action of the igdTs. The co-culture of igdTs and Jurkat cells at various E:T ratios showed the dosedependent cytotoxic effects of igdTs ( Figures 4A and S3E). Notably, even at an E:T ratio of only 0.25:1, cytotoxicity was clearly observed and reached a plateau at 2:1, while a previous report on iPSC-derived T cells showed their cytotoxicity toward lymphoma cell lines at E:T ratios of greater than 20:1 (Themeli et al., 2013). To evaluate the persistence of cytotoxicity, we co-cultured igdTs and Jurkat cells at a low E:T ratio of 0.5:1 for up to 4 days. The results showed that cytotoxicity increased in a time-dependent manner and lasted for at least 4 days ( Figures 4B and S3F).
Next, we investigated the mechanism by which igdTs exhibit cytotoxicity. Both blocking antibodies for gdTCR and NKG2D reduced the cytotoxicity of the purified igdTs, suggesting that the igdTs recognize tumor cells by both gdTCR and NKG2D ( Figure 4C). Perforin, granzyme B, and interferon gamma (IFNg) are reported to play important roles in the cytotoxicity of gdT cells (O'Neill et al., 2020). To determine whether igdTs release these factors, igdTs re-generated from the 62B3 gdT-iPSC line and Jurkat cells were co-cultured with the addition of Brefeldin A, which blocks the transportation of proteins to the Golgi bodies and induces the accumulation of proteins in the ER. The igdTs were pre-labeled with a CD3 antibody before co-culture to distinguish igdTs from Jurkat cells. At 4 h of co-culture, the cells were fixed with 4% PFA and analyzed by FCM. Granzyme B and perforin, which are expressed in cytotoxic T cells (Brandes et al., 2009;Voskoboinik et al., 2015), were expressed in both the igdTs and PBgdT cells ( Figure 4D), suggesting that the igdTs, like authentic gdT, directly attach to and attack tumor cells with lytic granules carried by secretory lysosomes.
Notably, no igdTs were positive for IFNg. In contrast, most granzyme B-positive cells in PBgdT cells were positive for IFNg ( Figure 4D). This finding raised the question as to whether igdTs have other distinct molecular signatures from PBgdTs.
In general, T cells are divided into four subsets of naive or memory phenotypes corresponding to the CD45RA and CD27 expression patterns (Berard and Tough, 2002). Despite stimulation during the same period, most igdTs showed a CD45RA(+) CD27(À) phenotype. In contrast, PBgdTs existed in all four subsets ( Figure 4E bottom panels). CD45RA(+) CD27(À) gdT cells have been reported to correspond to terminally differentiated effector memory T cells, which have a low expansion capacity (Odaira et al., 2016). Although the significance of the expression patterns of CD45RA and CD27 in gdT cells remains unclear, the expression patterns of these molecules also differ between igdTs and PBgdTs.  scRNA-seq reveals distinct populations of gdT cells in PBgdT cells and igdTs gdT cells have been reported to have various subtypes (Lawand et al., 2017;Li et al., 2020;Wu et al., 2017) and it was found that igdTs and PBgdTs show different expression patterns in the bulk state from the verification of cell surface markers. To examine whether there are differences in the subtypes of gdT cells between igdTs and PBgdT cells, we performed targeted single-cell RNA sequencing (scRNA-seq) of the following three types of cells: (1) freshly isolated PBMCs (no stimulation and no sorting). (2) PBgdT cells; PBMCs were expanded with HMBPP in vitro for 7 days and CD3(+) gdTCR(+) cells were sorted. (iii) igdTs; differentiated cells from gdT-iPSC clone 62B3 (according to the protocol shown in Figure 1A) were stimulated with HMBPP for 6-12 days in three independent experiments and CD3(+) gdTCR(+) cells were sorted. Unsupervised clustering of three datasets (freshly isolated PBMCs, PBgdT cells, and one of three igdT samples) identified six distinct cell clusters, which was shown by t-distributed stochastic neighbor embedding (t-SNE) ( Figure 5A).
The t-SNE distribution of each sample and the fraction of clusters in each sample are shown in Figures 5C and  5D, respectively. As expected, freshly isolated PBMCs were mostly occupied by abT cells (cluster 4) and non-T cells (cluster 5) and contained 5.2%, 1%, and 1% of gdT subsets 1, 2, and 3, respectively ( Figure 5D left bar graph). In PBgdT cells, gdT subset 2 accounted for approximately 70% of the total with 16.3% of gdT subset 1 and 1.7% of gdT subset 3 ( Figure 5D middle bar graph). On the other hand, gdT subset 1 accounted for the majority (89.1%) in igdTs. The rest was gdT subset 3 at 2.8%, and gdT subset 2 was completely absent ( Figure 5D right bar graph). These results indicated that the major gdT subsets differ between PBgdT cells and igdTs, and that cells similar to the major subset of igdTs exist in PBgdT as a minor subset as well as in PBMCs.
Differentially expressed genes in each gdT cell subset Next, we extracted differentially expressed genes in each cluster against the rest of the clusters (e.g., cluster 1 vs. the mean of clusters 2-6). Cells in gdT subset 1, the main population of igdTs, were enriched for NK cell-related genes (e.g., CTSW, FCER1G, KLRC3, CD244, NKG7, as well as the cytotoxic marker perforin coding gene [PRF1]). Cells in gdT subset 2, the dominant population of in vitro-expanded PBgdTs stimulated with HMBPP, expressed immune checkpoint inhibitory receptors (PDCD1 [PD-1], CTLA4, and LAG3) ( Figure 5E). The dominant population of igdT were positive for the expression of these inhibitory receptor genes in cells in gdT subset 1, but the expression levels were lower than those in gdT subset 2. Antigen-presenting genes (CD74, HLA-DQB1, HLA-DMA, HLA-DPA1, HLA-DRA) and IFNg and IFNg-inducing genes (IL12RB, IRF4) (Xu et al., 2010;Yao et al., 2013) were expressed at higher levels compared with cells in other clusters. The  RORC expression was restricted to cells in gdT subset 3 ( Figures 6A and 6B left panel). IL-17A, which was reported to be released from RORC+ gdT cells (Ben Hmid et al., 2020), was not expressed in cells in gdT subset 3 or in any of the other cells in this study ( Figure 6B right panel). A violin plot showed that KLRC3 and LAG3 were specifically expressed in gdT subsets 1 and 2, respectively ( Figure 6C). The expression of KIT was higher in the cells of gdT subsets 1 and 3 ( Figures 6A and 6C). On the other hand, pan-T cell marker CD2 and MHC class II molecule HLA-DRA were expressed in gdT subset 2, but not in gdT subsets 1 and 3 ( Figures 6A and 6C).
In order to investigate the reproducibility of the igdT data, the expression of these marker genes in scRNA-seq data of igdTs prepared three times independently was shown by a heatmap. The similar expression patterns indicated that the gene expression of igdTs was reproducible ( Figure S4E).
Taken together, we successfully re-generated MHC-unrestricted cytotoxic gdT cells from iPSCs and clarified the distinctive molecular signatures of iPSC-derived gdT cells.

DISCUSSION
In the present study, we successfully re-generated CD3(+) gdTCR(+) cells from gdT cell-derived iPSCs. Although there have been reports of the re-generation of abT cells from abT cell-derived iPSCs (Maeda et al., 2016;Nishimura et al., 2013;Vizcardo et al., 2013), it was unclear whether a protocol similar to that for abT cell differentiation from iPSCs could be applied to gdT cells, because the development process of gdT cells was reported to differ from that of abT cells in several points. First, during fetal development, gdT cells precede abT cells (Hayday, 2000). HPCs first differentiate into CD4/8 double-negative (DN) cells and then progress to CD4/8 double-positive (DP) cells (Seo and Taniuchi, 2016). While abT cells differentiate from DP cells, gdT cells can differentiate from both DP and DN cells (Van Coppernolle et al., 2012). The weak and strong TCR signal strength received by DN cells favors abT and gdT lineage development, respectively (Hayes et al., 2005). Furthermore, transcription factor Bcl11b-knockout mouse studies revealed that Bcl11b was essential for the differentiation of DN cells into abT cells, but not necessary for differentiation into gdT cells (Ikawa et al., 2010), and these gdT cells without Bcl11b only show a CD5(À) phenotype (Hatano et al., 2017). The expression of Bcl11b and CD5 were low in our igdTs, as shown in Figures 4E and 5E, suggesting that the development of igdTs may be similar to that of CD5(À) gd T cells in vivo.
With a scRNA-seq analysis, we revealed the distinct signatures of igdTs from PBgdTs. They shared common clusters with a minor part of freshly isolated PBMCs and ex vivo-expanded PBgdTs, indicating that the cells (B) The proportions of live Jurkat cells after co-culture with or without 62B3-derived igdTs for 1 day (n = 8 independent experiments, mean ± SD, two-tailed paired t test). (C) Representative continuous images at 0 and 12 h displaying GFP-Huh-7 cells co-cultured with or without 62B3-derived igdTs. The proportion of the GFP-positive area in target cells co-cultured with igdTs for 12 h was 28.7% of the GFP-positive area of the target cells with no effector, which was set to 100%. See also Figure S3B. Scale bars indicate 100 mm. (D) The relative proportion of live GFP-Huh-7 cells after co-culture with or without igdTs was calculated according to the GFP-positive area. The proportion of the no-effector group at 12 h was set to 100%, as a control (n = 3 independent experiments, mean ± SD, two-tailed paired t test). (E) Representative continuous images at 0 and 16 h displaying CFSE-stained SW480 cells co-cultured with or without 62B3-derived igdTs. The proportion of the CFSE-positive area at 16 h in target cells co-cultured with igdTs was 64.4% of the CFSE-positive area of the target cells with no effector, which was set to 100%. Scale bars indicate 100 mm. (F) The relative proportion of live CFSE-stained SW480 cells after co-culture with or without igdTs was calculated by CFSE-positive area. The proportion of the no-effector group at 16 h was set to 100%, as a control (n = 3 independent experiments, mean ± SD, two-tailed paired t test). (G) Huh-7 cells were incubated with CD3(+) sorted igdTs or PBgdTs at an E:T ratio of 2:1 and the cytotoxicity was determined using an xCELLigence RTCA system. The proportion of the no-effector group was set to 0%, as a control (n = 3 independent experiments, mean ± SD, two-tailed paired t test). (H) K562 cells were incubated with CD3(+) sorted igdT or PBNK cells, and the cytotoxicity was determined using an xCELLigence RTCA system. The proportion of the no-effector group was set to 0%, as a control (n = 3 independent experiments, mean ± SD, two-tailed paired t test). (legend continued on next page) resembling the major population of igdTs exist in adult PBMCs in nature but are not expandable with HMBPP stimulation, at least under the culture condition used in this study. Previously there have been many studies concerning the classification of human peripheral gdT cells. For example, the functions of gdT cells were reportedly separated into five subsets: IFN-g-producing, antigen-presenting, follicular B helper, regulatory gdT, and IL-17-producing cells (Wu et al., 2017). One other group classified gdT cells according to the effects on tumor cells: anti-tumor or tumor promoting . Another group divided gdT cells according to the expression of activation marker genes, such as CD16 (Braakman et al., 1992), CD69 (Cibrian and Sanchez-Madrid, 2017), and RORC (Ben Hmid et al., 2020). Our igdTs did not fully correspond to any of these previously reported gdT cell types in postnatal peripheral blood, in terms of the gene expression patterns.
Notably, in contrast to PBgdTs, the igdTs were negative for CD2 and positive for CD7. A previous report showed that a human fetal thymus-derived gdT cell clone showed a CD2 (low) CD7(+) phenotype and low IFNg secretion (Carding et al., 1990). Our igdTs might correspond to gdT cells derived from the fetal thymus. The cytotoxicity of our igdTs, which showed distinctive molecular signatures, can be supported by some previous reports. The igdTs were CD5(À), and CD5(À) gdT cells were reported to be more cytotoxic than CD5(+) gdT cells (Srour et al., 1990). In addition, approximately three-quarters of the igdTs showed a terminally differentiated T cell phenotype: CD45RA(+)/CD27(À). Terminally differentiated g9d2T cells reside in inflamed tissue, where they display an immediate effector function (Dieli et al., 2003) and exert higher cytotoxicity and lower IFNg production compared with other subsets in terms of the CD45RA and CD27 expression pattern (Caccamo et al., 2005). Together, molecular mechanisms that link the molecular signatures of our igdTs and their function should be clarified in future studies.
NK cell-related markers were expressed in igdTs. A subset of authentic gdT cells reportedly expressed NK cell-related genes and recognize target cells by a similar mechanism to NK cells. In addition, it is reported that mimetic-gd NKT cells, which expressed low T cell-related genes and high NK cell-related genes, were induced from iPSCs (Zeng et al., 2019). The shared NK cell markers support tumor direct recognition by gdT cells in an MHC-unrestricted manner (Wrobel et al., 2007). This NK-related gene expression may be responsible for the cytotoxicity of igdTs.
We herein demonstrated that our igdTs were completely negative for abTCR and that they killed tumor cells in an MHC-independent manner. The negative expression of abTCR may reduce the risk of graft-versus-host disease (Radestad et al., 2014). For this reason, there were studies in which allogenic PBgdTs were used as carriers for chimeric antigen receptor T (CAR-T) (Rozenbaum et al., 2020) and TCR-T (Ichiki et al., 2020).
Several limitations of the present study should be addressed in our future studies. First, the induction efficiency of igdTs was not satisfactory, and we have not clarified what CD3(À) cells existing after igdTs induction were. Second, it should be evaluated whether or not the igdTs attack noncancer cells of KIR-ligand mismatch recipients. Third, the igdT induction protocol established in this study used xenogenic serum and feeder cells, which are difficult for clinical applications. We are currently trying to generate igdTs under feeder-free and serum-free conditions (data not shown). Our technologies will advance off-the-shelf gdT cell-based immune therapy.

Materials availability
This study did not generate new unique reagents.

Data and code availability
The accession number for the scRNAseq reported in this paper is GEO: GSE194072.

Differentiation to gdT cells from iPSCs
Seven days before induction, human gdT-iPSCs were seeded onto a six-well plate at a density of 2.0 3 10 3 cells and cultured in StemFit medium (Ajinomoto, AK02N). On day 0, the medium was completely replaced by StemFit medium supplemented with 4 mM CHIR99021 (Tocris, 4423), 80 ng/ mL BMP4 (R&D, 314-BP), and 80 ng/mL vascular endothelial growth factor (VEGF) (R&D, 293-VE). On day 2, the medium was replaced by Essential 6 medium (Thermo Fisher, A1516501) supplemented with 2 mM SB431542 (WAKO, 033-24631), 50 ng/mL bFGF (WAKO, 060-04543), 50 ng/mL SCF (R&D, 255-SC), and 80 ng/mL VEGF. On day 4, the medium was replaced by StemPRO-34 SFM (Thermo Fisher, 10639-011) supplemented with 2 mM L-glutamine, 50 ng/mL IL-3 (Peprotech, AF-200-03), 50 ng/mL IL-6 (R&D, 206-IL), 50 ng/mL FLT3L (R&D, 308-FK), (C) The percentage cytolysis of Huh-7 cells after 12 h of co-culture with or without neutralizing antibodies (20 mg/mL). The percentage cytolysis of the no-effector group was set to 0%, as a control (n = 3 independent experiments, mean ± SD). (D) Flow cytometry to detect cytotoxic molecules. PBgdTs and igdTs were pre-incubated with Brefeldin A and co-cultured with Jurkat cells at the E:T ratio of 2:1 for 4 h. In the upper panels, effector cells were pre-labeled with a CD3 antibody before the start of co-culture. (E) The expression of T cell-related markers were analyzed by flow cytometry in gdT-iPSC-derived igdTs and PBgdTs. The cells were stimulated with HMBPP and IL-2 for 10 days and sorting was not performed.  (legend on next page) Kirin). On days 6 and 8, the medium was replaced with StemPRO-34 SFM supplemented with 2 mM L-glutamine, 50 ng/mL IL-6, 50 ng/mL SCF, and 10 IU/mL EPO. On day 10, hematopoietic cells were transferred into wells cocultured with feeder cells. Floating cells and supernatant were collected in the tube. Adhesive cells were dissociated with Accutase (Nacalai Tesque, 12679-54), and incubated at 37 C for 10 min. Supernatant was returned to the well, pipetted, and filtered using a 35-mm cell strainer. After centrifugation at 1,200 rpm for 4 min, cells were suspended in OP9 medium supplemented with 10 ng/ mL SCF, 10 ng/mL TPO (Peprotech, AF-300-18), 5 ng/mL IL-7 (R&D, 207-IL), 5 ng/mL FLT3L, and 100 mg/mL L-ascorbic acid (Nacalai Tesque, 30264-56). Cells were reseeded to the same well and incubated at 37 C for 30 min. Without pipetting, supernatant and floating cells were transferred into a new well confluent with pre-seeded OP9/N-DLL1 cells. On day 12, half of the medium was changed and cells were transferred into new wells with fresh OP9/N-DLL1 cells by vigorous pipetting. Then, half of the medium was changed every other day and cells were transferred onto fresh OP9/N-DLL1 cells every 6 days. On day 30, we collected cells with Accutase, similarly to day 10. Cells were suspended with RPMI1640 medium supplemented with 10% FBS, 1 nM HMBPP (Cayman Chemical, 13580), 100 IU/mL IL-2 (Shionogi Pharmaceuticals, Imunace), and 10 mM 2-mercaptoethanol and seeded onto new plates in a feeder-free condition. Half of the medium was changed every other day. After more than a week of stimulation, cytotoxicity was analyzed.

scRNA-seq
The day before the targeted scRNA-seq analysis, CD3(+)gdTCR(+) cells were sorted on a BD FACS Aria III from PBgdTs and igdTs that were stimulated with HMBPP for the indicated days as described above. To infer the origin of the sample, all cells were labeled with multiplex sample tags. Single-cell capture and cDNA library preparation were performed using a BD Rhapsody Single-Cell Analysis System with a BD Human Single-Cell Multiplexing Kit (BD Biosciences, #633781) and BD Human Immune Response Targeted Panel for Human (BD Biosciences, #633750), which contains 399 primer pairs, targeting 397 different genes, according to the manufacturer's recommendations. The concentration, size, and integrity of the resulting PCR products were assessed using a Qubit High-Sensitivity dsDNA Kit. Sequencing was performed using an Illumina HiSeq X (Illumina, San Diego, CA) in Macrogen (Tokyo, Japan). Fastq files were uploaded to the Seven Bridges Genomics online platform. The obtained counts were adjusted by distribution-based error correction (DBEC), an error correction algorithm developed by BD Biosciences. DBEC data were then loaded into Seurat (version 4.0.4.). Cells were then clustered using a resolution of 0.03 and visualized by t-SNE. The Seurat functions FeaturePlot, DotPlot, DoHeatmap, and Vlnplot were used to visualize the gene expression with feature plot, dot plot, heatmap, and violin plot, respectively. Markers for a specific cluster against all remaining cells were found by using the Seurat function FindAllMarkers. ScRNA-seq data have been deposited in GEO under accession number GSE194072.

Statistical analysis
Data are expressed as the mean ± SD. Differences between two groups were analyzed using a paired t test. Statistical analyses were performed using Microsoft Excel 2013 and EZR. p values of <0.05 were considered statistically significant.